Cellular regulatory mechanisms which rely on small dsRNA molecules (RNA silencing) are major (new) discoveries in biology.In many eukaryotic organisms silencing is achieved post-transcriptionally through pathways where dsRNA is synthesized by RNA-dependent RNA polymerases (RdRPs) and processed into 21-25 nucleotide long RNA molecules.Here we study QDE-1, an RdRP involved in the quelling (RNA silencing) pathway of Neurospora crassa. This filamentous fungus displays noticeable genomic stability, which has been attributed to quelling and other silencing mechanisms. Recently it was shown that QDE-1 is more active as a DNA-dependent RNA polymerase (DdRP) than as an RdRp and that it is also involved in DNA damage response (1,2). Recombinant QDE-1 displays five different enzymatic activities in vitro (3). The structural and biochemical data on this enzyme is extensive (1-5).We are interested on the basic biophysical parameters of QDE-1 action. The approaches taken rely on single molecule assays using high resolution optical tweezers, combined with fluorescence imaging. An optically levitated ‘dumbbell’ assay is used: the nucleic acid (NA) construct features one or two biotinylated ends that tether two different microspheres. The protein of interest is attached to the microsphere or directly to the NA tether. Since in our double-trap instrument one trap is stable whereas the other mobile, we can manipulate the tethers, detect changes in tether length and stiffness, and apply different forces and simultaneously observe the mobility of the fluorescently labelled template or protein.1. Lee, H-C et al. (2009). Nature 459(7244), 274-277.2. Lee, H-C et al. (2010). PLoS Biol. 8 (10), e1000469.3. Aalto et al. (2010). The Journal of Biological Chemistry, 285, 29367-29374.4. Salgado et al. (2006). PLoS. Biol. 4 (12), e434.5. Makeyev E.V. & Bamford D.H. (2002). Mol Cell 10(6), 1417-1427.